The need to visualize proteins that are suspended in a suspending medium or matrix or dissolved in a solution or to make proteins optically detectable in general is critical to a wide range of analytical, separatory, and diagnostic procedures. Electrophoretic separations are prominent examples of procedures that rely on protein detection, and electrophoresis in its various forms finds wide use in research laboratories and clinics and in on-site monitoring of water and other environmental media and biological samples. Among the various methods of making proteins optically detectable, the attachment of fluorescent markers is particularly useful, since the signals that these markers generate are readily quantifiable and can be read, analyzed, and recorded by automated detection equipment. Fluorescent signals also allow the user or the instrument in which they are generated to control the timing and intensity of the emitted signals.
In a typical biological sample, proteins are accompanied by a range of non-protein species such as nucleic acids, polysaccharides, lipids and other small molecules. Additional non-protein components that may also be present in the suspending medium are detergents, viscosity-control agents, buffers, carrier ampholytes, reductants, and unreacted monomers. The most effective means of applying a fluorescent marking to proteins in a thorough and reliable manner that is relatively free of background signals is the attachment of a fluorescent dye to the proteins while avoiding attachment of the dye to non-protein components. To confine the dye to proteins, non-protein interfering substances can be separated out prior to application of the dye, using for example the procedure described by Das et al. in U.S. Pat. No. 5,900,376, issued May 4, 1999. Unfortunately, procedures of this kind are time-consuming, labor-intensive or incomplete. The dye can also be covalently bonded to the proteins, but the covalent bonding itself raises concerns of selectivity relative to competing reactions with other substances in the medium, in addition to concerns of reactivity and the need to assure that the reaction is complete.
In analytical procedures in which the proteins are separated from each other in a matrix such as a polyacrylamide gel for purposes of detection, the proteins once separated are typically visualized and quantified by applying a solution of a dye that will selectively bind to the proteins or that will become fluorescent or visible upon association with protein. Doing this effectively generally requires extensive washing of the matrix to remove interfering substances such as detergents, buffers, and carrier ampholytes before the dye is applied. After application of the dye, further washing is required to remove unreacted or unbound dye.